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Evaluation of the FRTL-5 Cell Line to Further Confirm Potential Sodium Iodide Symporter (NIS) Inhibitors Identified by High Throughput Screening (HTPS)
Citation:
Devane, D., A. Buckalew, S. Laws, A. Murr, J. Wang, C. Deisenroth, AND T. Stoker. Evaluation of the FRTL-5 Cell Line to Further Confirm Potential Sodium Iodide Symporter (NIS) Inhibitors Identified by High Throughput Screening (HTPS). CHHE 4th Annual Symposium- Exploring Interactions Between the Brain and the Environment, Raleigh, NC, February 20, 2020.
Impact/Purpose:
Previously, our laboratory identified chemicals in the ToxCast libraries for their ability to inhibit the uptake of iodide by the sodium iodide symporter, which is necessary for thyroid hormone synthesis by the thyroid gland. Here, we used a secondary assay to confirm the most potent inhibitors.
Description:
Iodide uptake in the thyroid via the sodium-iodide symporter (NIS) is the first step in the biosynthesis of thyroid hormones. Thyroid hormones T3 and T4 are essential for regulating metabolism, cardiovascular function, fetal and neonatal neurodevelopment. Potential NIS inhibiting chemicals were previously screened in our laboratory using a human hNIS-HEK293T-EPA (hNIS) cell line and a radioactive iodine uptake assay (RAIU). Approximately 1800 ToxCast ph1_v2 and ph2 chemicals were screened with the HTPS assay and ranked for potency and cytotoxicity. Using the Fischer rat thyroid follicular (FRTL-5) cell line which endogenously expresses NIS, we evaluated the FRTL-5 in the RAIU as a secondary screening tool to further clarify and prioritize potential NIS inhibitors. Following assay validation, results from controls, reference and chemical tests indicated a highly robust and reproducible secondary assay. Test chemicals included; 30 top-ranked ToxCast chemicals from hNIS HTPS and 10 additional chemicals, which were then tested in parallel FRTL-5, hNIS, and Cell Titer Glo cell viability assays. An orthogonal propidium iodide cytotoxicity assay was also conducted to identify effects on cell membrane integrity. Test chemicals were run at six concentrations (0.001-100µM) in 3 independent biological replicates. The majority of the test chemicals showed IC50 values within one order of magnitude for NIS inhibition. These data demonstrate the utility of a secondary RAIU assay in a more physiologically relevant cell line that also provides a species comparison and in turn, strengthens the confidence for further prioritization. Several chemicals displaying strong NIS inhibition in both the FRLT-5 and hNIS cell lines with minimal cytotoxicity will be moved forward for additional testing in short-term in vivo assays, these include etoxazole, methoxyfenozide, cyprodinil and oxyfluorfen. This abstract does not necessarily reflect EPA policy.